Antibacterial Activity of Metallic Nanoparticles Antibacterial Activity of Metallic Nanoparticles

Metallic nanoparticles (NPs) for medical applications have been documented since ear- lier times. Advancement in modern medicines results in an increase in utilization of NPs for medical purposes due to their antibacterial, antiviral, antifungal, anti-inflammatory, and antiangiogenic properties. In this chapter, three metallic NPs are studied extensively as powerful nanoweapons for the destruction of bacteria. Recent research gives evidence that metallic NPs are very effective in supporting antimicrobial activities. The chemi cally and laser-ablated silver, gold, and copper NPs exhibited enhanced antibacterial activity than previously reported. The antibacterial mechanism was found dose- and size-dependent and was more profound for Gram-negative bacterium as compared to Gram-positive ones. The dose calculations of minimum inhibitory concentration (MIC) with NPs have been calculated for both Gram-positive and Gram-negative bacteria. The maximum zone of inhibition by disk diffusion was also experimented against various bacteria. These NPs exhibit excellent performance physically, catalytically, and chemi - cally. Present study will be beneficial in areas of environment, information technology, health, cosmetics, and food department. This chapter will cover the details of fabrication and antibacterial activity results of silver, gold, and copper NPs. This chapter endeavors to demonstrate the use of metallic NPs as an alternative antibacterial nanobiotics.


Introduction
Recently, metallic nanomaterials have become the most enormous and rapid emerging materials of science areas. The increased attention of nanomaterial fabrication, especially nanoparticles (NPs), is due to their fascinating properties revealed by their size, high surface area, and extraordinary surface activity exhibiting outstanding catalytic, electrical, and optical properties. Thus, metallic NPs have participated in extensive applications of research methodology medium can be well controlled [20][21][22] by adjusting the target, liquid type, and laser parameters, like wavelength, fluence, and pulse duration [23].

Wet chemical method for nanoparticle production
The second traditional method is wet chemistry mostly utilized to fabricate metallic NPs. This technique also has the ability to produce NPs of controlled morphology, composition, and crystallinity. Here, chemical reduction of salts generates comparatively larger NPs as compared to that of LAL technique [24,25]. In wet chemical method, the formation of NPs has undergone by the following processes: (A) Nucleation: The main process of synthesis initiates with the process of nucleation, in which a new phase particle called "seed" has been formed in a previous system of single phase (a homogenous solution of salt) [26]. With the additional attachment of metal atoms to this seed from solution, further growth of NPs is done. The shape of the NPs and its growth depends on the varying conditions of reaction [27][28][29][30][31].
(B) Seeding process: During the reduction process of salt, the metallic atom concentration enhances with the decomposition of precursor till a supersaturated state has been reached [32]. At this stage, the atoms begin to agglomerate and form nuclei of precise dimension called seed which further grow into crystallite [32]. Typically, precursors are metallic salts, mostly used as reducing agents [25,32]. Capping agents are used to stabilize the metallic seed by preventing agglomeration and maintaining the nuclei size [33]. Seed-mediated nuclei growth process is one of the most efficient and effective processes because of low activation energy requirement for metal reduction process on to the preformed seeds to get NPs of controlled size and shape [34,35].
(C) Nanoparticle formation: The nanoparticles are formed by the controlled competition of surface energy enhancement and a bulk energy diminution. An enhancement in the surface energy helps in dissolution, while decrease in bulk energy favors the process of growth [32]. Continued addition of atoms allows the seed to increase in size in a uniform manner. Thus, solutions of suitable metallic precursor, reducing agent and capping agent, are the basic necessity for the formation of metallic nanoparticles via chemical reduction method.

Antimicrobial mechanism of metallic NPs
Metal and metallic salts are well known for antibacterial mechanism for centuries as silver pots were used for drinking water from 4000 BCE [36,37]. Recent research in nanophysics capable the scientist to study the antibacterial properties on various metallic NPs [38,39].
Basic toxic antibacterial mechanism of metallic NPs is still under debate, but three main mechanisms are supposed which include, firstly, the formation of reactive oxidative species (ROS); secondly, releasing process of ions; and, finally, interaction of NPs with the cell membrane (Figure 1). Metallic NPs as compared to their salts have enhanced potential to combat bacterial infections [40][41][42][43]. Mostly, the size of NPs influences the antibacterial mechanism [44][45][46][47][48][49].

Entering the cell
The first step of antibacterial mechanism is the metallic ions of nanometer range attached to the cell via transmembrane protein. After attaching to bacterial cells, producing structural changes in the cell membrane and blocking the transport channels [6,50], the whole process is size dependent. Small NPs are more efficient, while larger NPs have a higher absolute surface area permitting for better adhesion property of van der Waals forces. Then, NPs may be internalized, produce ionization within the cell, and damage intracellular structures resulting in cell death (Figure 1) [40].

Reactive oxidative species (ROS) generation
The production of reactive oxidative species (ROS) by metal NPs plays a large role in their antibacterial effectiveness (Figure 1). ROS consist of short-lived oxidants, such as superoxide radicals (O −2 ), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (OH −1 ), and singlet oxygen (O −2 ) [51,52]. Due to the high reactivity of these species, ROS can cause damage to peptidoglycan and cell membranes, DNA, mRNA, ribosomes, and proteins [42]. ROS can also inhibit transcription, translation, enzymatic activity, and the electron transport chain [42,51]. Some metal oxide NPs rely on the generation of ROS as a main mechanism of toxicity [6,41,50].

Protein inactivation and DNA destruction
Metal atoms have the tendency to attach with thiol group of enzymes and finally deactivate the function of enzymes. It is also suggested that metal ions attach themselves between the pyrimidine and purine base pairs disturbing the bonding of hydrogen between two strands of antiparallel and destruct the molecule of DNA (Figure 1). Although this has to be further investigated, but this is true that metal ions have tendency to attach with DNA, once they go into the cell [53].

Detection methods
There are various direct and indirect methods for the microbial growth measurement. In direct method, mostly microbial effects are evaluated by viable technique of plate count, serial dilution, Figure 1. Various mechanisms of antimicrobial activity of the metal NPs [40]. and disk diffusion method, while indirect methods are analyzed by turbidity, dry weight, and by metabolic activity. In brief, short description of direct and indirect methods is as follows.

Viable plate count method
In the plate count techniques, Petri dishes are used for each reading. Agar plate is prepared, and one plate of inoculums and other plates with inoculum and nanoparticle solutions are spread on it with the help of sterile spreader; this plate is incubated for 24 hours at 37°C and then counted the colonies of each plate. Then, the inhibition percentage growth with each reading is calculated [54,55].

Disk diffusion method
The prepared culture of bacteria is mixed in nutrient broth to form liquid culture. Then, sterile nutrient agar solution is prepared and put into dishes and waited to be solidified. After that, holes are generated with the help of cork borer. Bacterial culture is spread on the agar with sterile cotton bud. Holes are filled with solution of metallic NPs and stabilized; after that, the plates are incubated for 24 hours at 37°C. The zones of inhibition are observed the next day and calculated with standard error. For accurate analysis, proper repetition of the experiments is done for microbial study [16].

Estimating bacterial numbers by indirect methods
Indirect methods are basically referred as time-consuming methods, and the large number of samples is prepared at a time. A spectrophotometer is utilized to analyze the turbidity process by measuring the quantity of light that transmits through a bacterial cell suspension. As more transmission occurs through suspension, it means that turbidity decreases, indicating the reduction in bacterial cells and vice versa [56,57].

Antibacterial mechanism of silver nanoparticles (SNPs)
In recent research, Zafar et.al. [57] have worked on the antibacterial mechanism of silver NPs fabricated by laser ablation and by chemical method having different sizes and examined these NPs against Gram-positive (+) bacteria (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (−) bacteria (Salmonella, Escherichia coli, and Klebsiella pneumonia) by turbidity method and by well diffusion method.

Turbidity method
A medium (nutrient broth) (5.00 ml) was arranged in the labeled tubes and then sterilized, thus forming broth media in transparent form. The bacterium culture was then ready by shifting an identified Gram-positive [S. aureus and B. subtilis] and Gram-negative bacteria's culture [Salmonella and E. coli] into four (4) tubes of liquid nutrient broth with platinum wire. After the process of inoculation, tubes were incubated at 37°C for 1 day. The next day, a visible growth was observed in the tubes of culture. Then, these tubes of bacterium culture were strictly checked by various identification tests. Already measured doses (low, medium, and high) of sterilized SNPs were tested against this bacterium growth. Each tube had facilitated with medium (5 ml), inoculums (200 μl), plus the measured dose of SNP stock solution. Three tubes were arranged for each reading. Negative control tube had broth and NPs only, and positive control tube had broth and inoculums only. All tubes were incubated at 37°C for 24 hours.
Turbidity of tubes was confirmed by Elisa reader, a spectrophotometer having a filter of 600 nm wavelength. The values of optical density (OD) from Elisa reader in absorption mode represented the bacterial growth of labeled samples. Three OD values of each dose were taken, and their mean was calculated with standard deviation (St. Dev.). Three designed doses of SNPs were taken. Bacterial growth in nutrient broth revealed the total percentage (%) of bacterium growth. The percentage (%) change in the growth of bacterium was measured against OD value of pure nutrient media of broth (reference OD value). Similarly, the maximum calculated growth of S. aureus, B. Subtilis, Salmonella, and E. coli was 75.19, 68.5, 74.3, and 71.9%, respectively [57]. Contaminant in microorganism's growth was only 1.5% which is ignorable against growth of bacterium ( Table 1).
Same process had been applied for the calculation of all doses. The growth of bacteria decreases with increasing dose of NPs [57]. Chemically synthesized SNPs inhibited 67.02% S. aureus, whereas E. coli was inhibited up to 87.9%. It indicated that more doses should design for full inhibition of S. aureus. B. subtilis was inhibited 39.9%, and Salmonella was inhibited only 80.2%. Laser-ablated SNPs inhibited the bacterium with higher efficiency having low dose (2.10 μg/ml). For example, 67.02% growth calculated with dose (2.35 μg/ml) for S. aureus diminished up to 92.0% with dose (2.10 μg/ml), whereas no growth appeared for E. coli with designed high doses. Similarly, the calculated growth of Salmonella is 18.0% that shows that the growth is reduced up to 82% [57].
Two sized SNPs were considered for antibacterial evaluation. The chemically synthesized SNPs have size range ~30-40 nm (Sample S1), while the laser-ablated SNPs had size range ~20-30 nm (Sample S2). Equal doses of laser-ablated NPs provided enhanced inhibition against each bacterium. Chemically synthesized SNPs exhibit less efficiency with respect to laser ablated SNPs. The first reason was slightly larger-sized nanoparticles, and, secondly, it might be possible that some chemical species may be adsorbed on the surface of NPs which declined the antibacterial performance. Hence, for the pureness of sample, chemical fabrication has many restrictions to employ these NPs for the biological, catalytic, and sensing applications.
For statistical analysis and regression line, along X-axis, dose of SNPs was taken, whereas along Y-axis, percentage growth was plotted. Regression line has been plotted by using the values from Table 2 in given equations: From regression lines (Figure 2)

Well diffusion method for silver nanoparticles
Antibacterial mechanism was further evaluated against S. aureus and E. coli with same doses of SNPs by well diffusion method [57]. The results [57] obviously determined the zone of inhibition for laser-ablated and chemically synthesized SNPs, and no zone appeared in the control. Further, the inhibition zones due to laser-ablated NPs are considerably enhanced as compared to chemically synthesized SNPs as shown in Figure 3 and Table 3.
In previous studies, Laszlo Korosi fabricated SNPs by LAL method and concluded that size of SNPs strongly effected on the antibacterial mechanism. The antibacterial activity of SNPs   (3 nm) was very much high against E. coli and SNPs (20 nm) revealed low antibacterial performance even though with concentration of 5 mg/L. However, S. aureus was partially with stand SNPs (3 nm) up to 5 mg/L [58]. In present study, SNPs give the best maximum zone (~ 15 and 23 mm for S. aureus and E. coli).

Antibacterial mechanism of gold nanoparticles
The antibacterial efficiency of two samples of chemically synthesized gold nanoparticles (GNPs) was evaluated by Shamaila et.al. [59]. The first sample of GNPs, G1, has a size range of ~ 6-34 nm, and the second sample, G2, has a size range ~ 20-40 nm. Their potential efficacy was checked against Gram-negative bacteria (E. coli, K. pneumonia) and Gram-positive bacteria (S. aureus, B. subtilis) by the method of turbidity and well diffusion technique [59].

Turbidity method
Same process was used for turbidity method as described in Section 2.  Table 3. Zone of inhibition for low, medium, and high doses of chemically synthesized and laser-ablated SNPs on S. aureus and E. coli [57].
Antibacterial Activity of Metallic Nanoparticles http://dx.doi.org/10.5772/intechopen.72526 The comparison of antibacterial results of two sets of samples of NPs having different sizes is given in Table 4. Equal doses of these samples were taken which give inhibition for each human pathogen. Sample G1 NPs exhibited more antibacterial mechanism with respect to sample G2 NPs due to size difference. There is difference in the antimicrobial results of GNPs for Gram-negative and Gram-positive bacterium. This is due to the difference in its membrane structure, i.e., the thickness of layer peptidoglycan and wide variation range between both types of bacteria [58]. Thus, inhibition of Gram-positive bacteria is achieved with higher dose of NPs.
Regression line has been plotted between doses and percentage (%) growth, exactly like silver NPs with given equations in Section 2.1.1. Curves of growth inhibition were plotted for G1 and G2 NPs. From Figure 4 [61,62]. However, in the current project, GNPs reveal the best performance, and maximum zone was observed with dose measurement against human pathogens.

Well diffusion method of gold nanoparticles
The doses were further tested to examine the zone of inhibition of S. aureus and E. coli by well diffusion method. Agar plates were used to evaluate the antibacterial action of GNPs against human pathogen bacteria E. coli and S. aureus, with same method as described for SNPs. The suspension of NPs (5, 15, and 30 μl) was poured into wells named 2, 3, and 4. The inhibitory zones were measured after incubation at 37°C for 24 hours.
G1 NPs and G2 NP results from method of well diffusion against E. coli and S. aureus bacteria are given in Figure 5 and in Table 5, where inhibition zones for GNPs are obvious. The inhibition zone of G1 NPs is larger than G2 NPs as confirmed (  Table 4. Antibacterial effects of two sized G1 and G2 GNPs against four bacteria [59].
Bacterial Pathogenesis and Antibacterial Control

Antibacterial mechanism of copper nanoparticles
In the present research of Khalid et al. [64], copper nanoparticles (Cu NPs) were used to evaluate the antibacterial mechanism. The size of chemically synthesized Cu NPs without any stabilizer was rapidly increased. Antibacterial mechanism of laser-ablated Cu NPs (with filter and without filter of MCE membrane) was examined against E. coli and S. aureus by the turbidity and disk diffusion method [24].

Turbidity method
Three concentrations of Cu NPs were used. Cu NPs were proved to be very toxic for bacteria E. coli and S. aureus. Three factors are important in the toxicity of Cu NPs such as concentration of NPs, particle size, and bacterial growth [64,65]. Thus, those experimental conditions were selected so that they exhibited the best curve of dose-response.  Table 6.
To find out how much maximum dose was required for complete reduction in the bacterial growth, statistical analysis was calculated by the equations given in Section 2.1.1. Figure 6 represents regression line.
By chemical reduction, Cu NP doses required for maximum growth inhibition of E. coli calculated by regression line were 6.07 μM for with membrane and 8.33 μM for without membrane. Similarly, maximum growth inhibition for S. aureus was 8.32 μM with membrane and  11.76 μM without membrane. The significant enhancement in concentration of NPs attributes higher toxicity and reduction in percentage growth of bacteria to minimum values.

Well diffusion method of copper nanoparticles
The zone of inhibition of laser-ablated Cu NPs was further observed [24] by agar method of well diffusion ( Table 7). The calculations of zone of inhibition for E. coli and S. aureus are for high doses 28 ± 0.01 mm and 21 ± 0.02 mm, respectively [24]. No zone of inhibition is observed for positive control (Figure 7).

Antibacterial mechanism of metallic nanoparticles
Antimicrobial performance of metallic NPs depends on the features of bacterial species. The main dissimilarity between Gram-negative and Gram-positive bacteria is its membrane structure such  that peptidoglycan layer thickness has the main features of polymer along with amino acid and sugar, thus forming an outside layer of plasma membrane (cell wall) which offers the stability to structure which is responding to the osmotic pressure of cytoplasm. Its range of thickness is higher (50%) in Gram-positive (+) and lower (8%) in Gram-negative (−) bacterium. Grampositive bacterium comprises peptidoglycan layer in multiple steps having long teichoic acid chain. Conversely, Gram-negative bacteria contain a single and thin peptidoglycan layer with the absence of teichoic acid. Outer membrane has periplasmic and lipopolysaccharide layer [58].

With MCE membrane growth inhibition (%) Without MCE membrane growth inhibition (%)
Thus, the main process is first the attachment of metal ions or NPs to the bacterium outer cell wall and then accumulation of protein precursor's layer, which ultimately disables the proton motive force. Metallic NP destabilizes the outer membrane, produces crack in the membrane of plasma, and reduces the synthase activities of the depletion layer of intracellular adenosine triphosphate (ATP) [31], thus reducing the process of metabolism. It destructs the ribosome subunit by t RNA binding and thus finally the total collapse of biological mechanism.
It has been further concluded that the small-sized metallic NPs with larger surface area exhibited some electronic effects which were beneficial to improve the surface attraction of NPs. In addition, the percentage of enhanced surface area straightly interacted with the membrane of microorganism with vast extent and hence bridged an improved relation with bacteria. These two vital factors powerfully enhanced the antimicrobial action of the NPs with high surface Sr. no.  Table 7. Effects of copper NPs having low, medium, and high doses on E. coli and S. aureus [24]. area. Bacterial proteins (cell wall and cytoplasm) were accountable for the cell performance. These NPs disrupted the normal performance of protein function, which causes the cell's death. NPs basically interacted with soft bases having sulfur or phosphorus components. So, the sulfur of proteins and phosphorus belonging to DNA molecules are favorite attacking sites of NPs. Then, these NPs attached themselves to enzymes (thiol groups), i.e., nicotinamide adenine dinucleotide (NADH) dehydrogenases and destructed its respiratory chain by releasing of oxygen species, thus creating oxidative stress. Consequently, a major damage is occurred in the cell structures and lastly leads to cell loss [21,59].

Concentration of nanoparticles
Shrivastava et al. [66] have reported that the SNPs affected S. aureus in lesser amount. The bacterium B. subtilis has been considered a most powerful bacterium (Gram-positive). The reason is that it can stay alive easily in any rigid condition due to endospore formation (stressresistant) and its formation of DNA externally which can be produced by recombination with original DNA at its last stage and can tolerate under adverse situations. Salmonella also exhibits more resistance with respect to E. coli.
In the present research work, SNPs exhibited effective antibacterial behavior against E. coli bacterium with respect to other bacterium. S. aureus exhibited more resistance than E. coli; so, selected dose (high ~2.10 μg/ml) is quite enough for maximum inhibition of E. coli. Due to variation in peptidoglycan layer in Gram-positive bacteria, more doses of SNPs would be recommended for full inhibition of Salmonella and S. aureus. The laser-ablated SNPs have demonstrated and elevated antibacterial behavior due to small size and purity of NPs which may be beneficial in wood flooring, coatings, and cotton textile industries.
It is demonstrated that the small-sized GNP sample ~ 6-34 nm displays good bacterial performance than the second GNP sample ~ 20-40 nm. This advantage is by reason of its minute size, enhanced surface-area-to-volume ratio, and fine penetration power. Finally, the smallsized NPs effortlessly bind to the prominent parts of the outer membrane, so causing damages in structure, deprivation, and ultimately cell death. Various GNP doses displayed the best antibacterial action against E. coli with respect to other tested bacteria. Similarly, dose of 2.70 μg/ml was quite sufficient for K. pneumonia and for E. coli's complete inhibition. More NP doses were recommended for total inhibition of S. aureus and B. subtilis because of their difference of peptidoglycan layer. Here, B. subtilis had been considered the most resistive Gram-positive bacteria in the performed experimentation.
It is evident that for both Gram-positive and Gram-negative bacteria, the increase in the concentration of Cu NPs revealed the decrease in percentage growth. With high dose of Cu NPs, a noticeable antibacterial action (growth inhibition ~ 1.9%) was recorded for E. coli with filtered NPs (smaller in size); however, at the same time for nonfiltered dose (larger in size), the percentage growth decreases up to only 27%. Similarly, the percentage growth of S. aureus for filtered NPs decreased up to 31.8% and for non-filtered up to ~ 42.1% only. The clear difference in percentage (%) growth inhibition for both strains of bacteria was deeply attributed to the concentration of NPs and size of NPs. These outcomes concluded that the further doses of Cu NPs are required to show full antibacterial action against these bacterial strains. The cell wall of Gram-positive bacteria is composed of heavy layer of peptidoglycan (about ~ 20-80 nm), forming a three-dimensional structure of rigid tissues. This rigidity protects the cell wall, provides only fewer sites for Cu NPs, and does not allow to penetrate the NPs [24].
In current research, the observed antibacterial mechanism of all metallic NPs is tested on behalf of concentration and size of NPs. The basic mechanism is that how many metal ions are released because they attributed to their large surface-area-to-volume ratio. These ions directly interact with each bacterium's outer membrane. One possible justification is nanometer size pores of bacteria cell membranes that can give a pathway for the NPs [59]; secondly, the ions released by NPs may fasten to the cell wall of bacteria and crack it. Ions of NPs which are released produce radicals of hydroxyl that may deform the basic function of essential proteins and finally its DNA for the cell death [57][58][59].

Conclusion
The two techniques for fabricating metallic nanoparticles were compared here to specify a broad range of achievable characteristics. This aptitude to give variation in properties of controllable metallic nanoparticles would be eventually profitable for future research relaying on the antibacterial properties of nanoparticles. A direct assessment between metallic NPs generated either by wet chemical technique or LAL method will be supportive in finding the exact trends and mechanisms of antimicrobial, which are currently hot issues of recent advance research. The antibacterial activity was size and dose dependent and was more explicit against Gram-negative bacterium than Gram-positive ones. The fabricated nanoparticles are spherical in shape with polycrystalline nature having various size ranges in nm. Antibacterial behavior of metallic nanoparticles against pathogenic bacteria demonstrates that they can act as an efficient tool for antibacterial. Silver, gold, and copper nanoparticles exhibit their excellent performance physically, catalytically, and chemically because of its larger surface-area-to-volume ratio. Turbidity provides an efficient and fast technique for the estimation of the bacterial development in a liquid. The calculations of MIC are further supportive in calculation of inhibition zone by agar method of diffusion. The present study will be beneficial in environment, information technology, health, cosmetics, and food department fields. Additionally, silver nanoparticles have reduced the life-threatening facts because of their flexible nature with respect to conventional antibiotics. Gold nanoparticles provide benefit in the field of biomedicine, chemistry, and genetics due to their characteristics of functionally active power. In biomedicine, gold NPs can become a vital revolution in the research of drug delivery and cancer therapy. They also proved to be nontoxic and a safe antimicrobial representative due to their powerful functional nature instead of antibiotics. Antimicrobial nanomaterials are potentially eye-catching for a variety of medical applications, i.e., dental procedures. Copper nanoparticles perform best as an antibiotic, antifungal, and antimicrobial agent when utilized for plastics, textiles, and coatings. Copper and its oxide are used for the coating of cellulose of nanofibrillar, a promising nanobiomaterial. The metallic nanoparticles showed potential performance as an antimicrobial against E. coli, S. aureus, B. subtilis, and Salmonella and are suggested as an alternative antibacterial nanobiotics.
appreciation is given to the assistance of the Physics Department, University of Engineering and Technology, Lahore, Pakistan. Authors are highly obliged to the editors of Intech Open Book for providing this great opportunity.